Highly active antiretroviral therapy (HAART) is able to sustain suppression of viral replication in HIV-1 infected individuals. Nevertheless, viral replication rapidly rebounds if therapy is interrupted. The prevailing view is that long-lived, quiescent memory CD4+T lymphocytes contain latent proviruses which can rekindle viral replication if therapy is interrupted. However, we have evidence that HIV-1 replication persists in infected individuals on HAART and that episomal viral cDNA can be detected in the majority of patients on HAART. We demonstrate that episomal cDNAs are unstable in vitro and dynamic in vivo and, as such, are indicators of ongoing replication. The presence of episomal cDNA in gut associated lymphoid tissue (GALT) and lymph node (LN) of patients on HAART implicates lymphoid tissue (LT) as a reservoir for HIV-1 persistence in the face of therapy. This application is built on the following hypotheses: Hypothesis 1: HIV-1 replication persists in lymphoid tissue in patients on HAART and this reservoir of cryptic replication fuels viral recrudescence upon therapy interruption. Hypothesis 2: Cryptic replication is a consequence of subinhibitory drug concentrations in GALT and LN CD4+cells. Hypothesis 3: Cryptic replication compromises immune function in GALT and LN of patients on HAART. To investigate these hypotheses, we propose to: Aim 1: Determine whether covert replication persists in lymphoid tissue in the face of HAART and whether this replication fuels viremia upon treatment interruption. Viral envelope sequences within unintegrated (episomal) and proviral cDNA in GALT and LN CD4+ T cells and macrophages will be cloned and sequenced. Phylogenetic comparison of these envelope sequences with analogous sequences in recrudescing viral RNA following treatment interruption will be used to establish whether GALT and LN serve as reservoirs of cryptic replication in patients on HAART. Aim 2: Define how cryptic viral replication persists in the face of HAART and whether cryptic replication undermines immune function in patients on HAART. We will determine whether a correlation exists between the level of cryptic viral replication and concentrations of drug in lymphoid tissue of patients on HAART and whether the extent of cryptic viral replication correlates with the magnitude of the immune defect (CD4 cell number and HSV2 shed rates) in lymphoid tissues in patients on HAART. We believe that these studies will identify the mechanism of viral persistence during HAART and provide the rationale for new treatment strategies to more effectively truncate ongoing viral replication.